The protective effects of dezocine on interleukin-1β-induced inflammation, oxidative stress and apoptosis of human nucleus pulposus cells and the possible mechanisms

Abstract

Intervertebral disc degeneration (IDD) is a natural problem linked to the inflammation. We aimed to investigate the role of dezocine (DEZ) in the development of IDD. Human nucleus pulposus cells (HNPCs) induced by interleukin (IL)-1β was used as a cellular model of IDD. After treatment with DEZ, HNPCs viability was evaluated with a CCK-8 assay. Then, the levels of inflammatory factors, including IL-6 and tumor necrosis factor-α (TNF-α), and oxidative stress-related markers, including reactive oxygen species (ROS), malondialdehyde (MDA) and reduced glutathione (GSH), were tested by RT-qPCR or kits. TUNEL staining was employed to detect cell apoptosis and Western blot was used to determine the expression of proteins related to inflammation, oxidative stress, apoptosis, endoplasmic reticulum stress (ERS) and MAPK signaling. Afterward, PMA, a MAPK signaling pathway agonist, was adopted for exploring the regulatory effects of DEZ on MAPK pathway. Results indicated that DEZ enhanced cell viability of HNPCs after IL-1β exposure. DEZ alleviated the inflammation and oxidative stress, evidenced by decreased levels of IL-6, TNF-α, ROS, MDA, p-NF-κB p65, NF-κB p65 in nucleus, cox-2 and increased levels of NF-κB p65 in cytoplasm, GSH, SOD1 and SOD2. Moreover, DEZ notably inhibited IL-1β-induced apoptosis of HNPCs. Furthermore, DEZ suppressed the levels of ERS-related proteins. The levels of related proteins in MAPK signaling including p-P38 and p-ERK1/2 were remarkably reduced after DEZ administration. By contrast, PMA crippled the impacts of DEZ on inflammation, oxidative stress and apoptosis of HNPCs induced by IL-1β. Collectively, DEZ ameliorates IL-1β-induced HNPCs injury via inhibiting MAPK signaling.

Keywords: Intervertebral disc degeneration; dezocine; endoplasmic reticulum stress; human nucleus pulposus cells; inflammation; mapk.

Figure 1.

DEZ treatment elevated the viability of HNPCs after IL-1β exposure. (a) The chemical structure of DEZ. (b) Cell viability was examined with a CCK-8 assay after HNPCs treatment with different concentrations (0.5, 1, 2, 4 and 8 μg/mL) of DEZ. *P < 0.05 vs. 0 μg/mL. (c) Cell viability was determined with a CCK-8 assay when HNPCs were exposed to 0.5, 1 and 2 μg/mL DEZ under IL-1β stimulated condition. ***P < 0.001 vs. untreated group; ^#P < 0.05, ^###P < 0.001 vs. IL-1β group.

Figure 2.

DEZ decreased the inflammation and oxidative stress in IL-1β induced HNPCs. (a) The expression of IL-6 and TNF-α was assessed by RT-qPCR after treatment with various concentrations of DEZ in HNPCs treated with IL-1β. (b) ELISA was adopted to evaluate the contents of IL-6 and TNF-α. (c) The protein levels of p-NF-κB p65, p65 expression in nucleus, p65 expression in cytoplasm and cox-2 were examined with Western blot analysis. (d) The ROS content was tested by means of the 2ʹ, 7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. The levels of (e) MDA and (f) GSH were measured using commercial kits. (g) Western blot assay was utilized to determine the protein levels of SOD1 and SOD2. ***P < 0.001 vs. untreated group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IL-1β group.

Figure 3.

DEZ attenuated the apoptosis and ERS in IL-1β induced HNPCs. (a) Cell apoptosis was tested by TUNEL staining. (b) Western blot was executed for assessing the levels of apoptosis-related proteins. (c) The levels of ERS-related proteins were evaluated with Western blot assay. ***P < 0.001 vs. untreated group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IL-1β group.

Figure 4.

DEZ suppressed IL-1β-induced activation of MAPK signaling pathway in HNPCs. Western blot assay was applied to evaluate the protein levels of phosphorylated p38, p38, phosphorylated ERK1/2 and ERK1/2. ***P < 0.001 vs. untreated group; ^##P < 0.01, ^###P < 0.001 vs. IL-1β group.

Figure 5.

Inhibitory effects of DEZ on inflammation and oxidative stress of IL-1β induced HNPCs were restored by MAPK pathway agonist PMA. (a) The expression of IL-6 and TNF-α was assessed by RT-qPCR. (b) ELISA was utilized to evaluate the contents of IL-6 and TNF-α. (c) The protein levels of p-NF-κB p65, p65 expression in nucleus, p65 expression in cytoplasm and cox-2 was examined with Western blot analysis. (d) The ROS content was tested by means of the 2ʹ, 7ʹ-dichlorodihydrofluorescein diacetate (DCFH-DA) staining method. The levels of (e) MDA and (f) GSH were measured using commercial kits. (g) Western blot assay was utilized to determine the protein levels of SOD1 and SOD2. ***P < 0.001 vs. untreated group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IL-1β group; ^ΔP<0.05, ^ΔΔP<0.01, ^ΔΔΔP<0.001 vs. IL-1β+DEZ group.

Figure 6.

Inhibitory effects of DEZ on apoptosis and ERS of IL-1β induced HNPCs are diminished by MAPK pathway agonist PMA. (a) The apoptotic rate was tested with TUNEL assay. (b) The levels of apoptosis-related proteins were detected with Western blot. (c) The levels of ERS-related proteins were evaluated with Western blot assay. ***P < 0.001 vs. untreated group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IL-1β group; ^ΔP<0.05, ^ΔΔP<0.01, ^ΔΔΔP<0.001 vs. IL-1β+DEZ group.